Human studies suggest that a variety of prenatal stressors are related to high risk for cognitive and behavioral abnormalities associated with psychiatric illness (Markham and Koenig, 2011). Recently, a downregulation in the expression of GABAergic genes (i.e., glutamic acid decarboxylase 67 and reelin) associated with DNA methyltransferase (DNMT) overexpression in GABAergic neurons has been regarded as a characteristic phenotypic component of the neuropathology of psychotic disorders (Guidotti et al., 2011). Here, we characterized mice exposed to prenatal restraint stress (PRS) in order to study neurochemical and behavioral abnormalities related to development of schizophrenia in the adult. Offspring born from non-stressed mothers (control mice) showed high levels of DNMT1 and 3a mRNA expression in the frontal cortex at birth, but these levels progressively decreased at post-natal days (PND) 7, 14, and 60. Offspring born from stressed mothers (PRS mice) showed increased levels of DNMTs compared to controls at all time-points studied including at birth and at PND 60. Using GAD67-GFP transgenic mice, we established that, in both control and PRS mice, high levels of DNMT1 and 3a were preferentially expressed in GABAergic neurons of frontal cortex and hippocampus. Importantly, the overexpression of DNMT in GABAergic neurons was associated with a decrease in reelin and GAD67 expression in PRS mice in early and adult life. PRS mice also showed an increased binding of DNMT1 and MeCP2, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine in specific CpG-rich regions of the reelin and GAD67 promoters. Thus, the epigenetic changes in PRS mice are similar to changes observed in the post-mortem brains of psychiatric patients. Behaviorally, adult PRS mice showed hyperactivity and deficits in social interaction, prepulse inhibition, and fear-conditioning that were corrected by administration of valproic acid (a histone deacetylase inhibitor) or clozapine (an atypical antipsychotic with DNA-demethylation activity). Taken together, these data show that prenatal stress in mice induces abnormalities in the DNA methylation network and in behaviors indicative of a schizophrenia-like phenotype. Thus, PRS mice may be a valid model for the investigation of new drugs for schizophrenia treatment targeting DNA methylation. This article is part of a Special Issue entitled 'Neurodevelopment Disorder'.

DNA methylation is an epigenetic regulatory mechanism commonly associated with transcriptional silencing. DNA methyltransferases (DNMTs) are a family of distantly related proteins that both catalyze the de novo formation of 5-methylcytosine and maintain these methylation marks in cell-specific patterns in virtually all mitotic cells of the body. In the adult brain, methylation occurs in progenitor cells of the neurogenic zones and in post-mitotic neurons. Of the DNMTs, DNMT1 and DNMT3a are most highly expressed in post-mitotic neurons. While it has been commonly thought all post-mitotic neurons and glia express DNMTs at comparable levels, the co-expression of selected DNMTs with markers of distinct neurotransmitter phenotypes has not been previously examined in detail in the mouse. To this end, we analyzed the expression of DNMT1 and DNMT3a along with GAD67 in the brains of the GAD67-GFP knockin mice. After first confirming that GFP immunopositive neurons were also GAD67-positive, we showed that in the motor cortex, piriform cortex, striatum, CA1 region of the hippocampus, dentate gyrus, and basolateral amygdala (BLA), GFP immunofluorescence coincided with the signal corresponding to DNMT1 and DNMT3a. A detailed examination of cortical neurons, particularly in cortical layers III to V, showed that ∼30% of NeuN immunopositive neurons were also DNMT1 positive. These data do not exclude the expression of DNMT1 or DNMT3a in glutamatergic neurons and glia. However, they suggest that their expression is very low compared with the levels present in GABAergic neurons.

The clinical use of diazepam or midazolam to control organophosphate (OP) nerve agent-induced seizure activity is limited by their unwanted effects including sedation, amnesia, withdrawal, and anticonvulsant tolerance. Imidazenil is an imidazo-benzodiazepine derivative with high intrinsic efficacy and selectivity for α2-, α3-, and α5- but low intrinsic efficacy for α1-containing GABA(A) receptors. We have previously shown that imidazenil is more efficacious than diazepam at protecting rats and mice from diisopropyl fluorophosphate (DFP)-induced seizures and neuronal damage without producing sedation. In the present study, we compared the tolerance liability of imidazenil and diazepam to attenuate the seizure activity and neurotoxic effects of DFP. Rats received protracted (14 days) oral treatment with increasing doses of imidazenil (1-4 mg/kg), diazepam (5-20 mg/kg), or vehicle. Eighteen hours after the last dose of the protracted treatment schedule, rats were tested for anticonvulsant tolerance after a 30 min pretreatment with a single test dose of imidazenil (0.5 mg/kg) or diazepam (5 mg/kg) prior to a DFP challenge (1.5 mg/kg). The anticonvulsant (modified Racine score scale) and neuroprotective (fluoro-jade B staining) effects of diazepam were significantly reduced in protracted diazepam-treated animals whereas the effects of imidazenil were not altered in protracted imidazenil-treated animals. The present findings indicate that protracted imidazenil treatment does not produce tolerance to its protective action against the neurotoxic effects of OP exposure.

Nicotine improves cognitive performance and attention in both experimental animals and in human subjects, including patients affected by neuropsychiatric disorders. However, the specific molecular mechanisms underlying nicotine-induced behavioral changes remain unclear. We have recently shown in mice that repeated injections of nicotine, which achieve plasma concentrations comparable to those reported in high cigarette smokers, result in an epigenetically induced increase of glutamic acid decarboxylase 67 (GAD(67)) expression. Here we explored the impact of synthetic α(4)β(2) and α(7) nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α(4)β(2) and a lower affinity full agonist at α(7) neuronal nAChR, injected in doses of 1-5 mg/kg/s.c. twice daily for 5 days, elicited a 30-40% decrease of cortical DNA methyltransferase (DNMT)1 mRNA and an increased expression of GAD(67) mRNA and protein. This upregulation of GAD(67) was abolished by the nAChR antagonist mecamylamine. Furthermore, the level of MeCP(2) binding to GAD(67) promoters was significantly reduced following VAR administration. This effect was abolished when VAR was administered with mecamylamine. Similar effects on cortical DNMT1 and GAD(67) expression were obtained after administration of A-85380, an agonist that binds to α(4)β(2) but has negligible affinity for α(3)β(4) or α(7) subtypes containing nAChR. In contrast, PNU-282987, an agonist of the homomeric α(7) nAChR, failed to decrease cortical DNMT1 mRNA or to induce GAD(67) expression. The present study suggests that the α(4)β(2) nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α(7) nAChR agonists.

Schizophrenia postmortem brain is characterized by gamma aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.

Recent evidence suggests that alpha1-containing GABA(A) receptors mediate the sedative, amnestic, and to some extent the anticonvulsant actions of non-selective benzodiazepine (BZ) receptor ligands, such as diazepam (DZ). Anxiolytic and in part, anticonvulsant actions of BZ ligands are mediated by alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. This has resulted in increasing interest in developing BZ ligands with selective actions at GABA(A) receptors, including alpha2-, alpha3-, and alpha5-subunits, but devoid of efficacy at alpha1-containing receptors. To refine their spectrum of pharmacological actions, efforts are being made to minimize unwanted effects such as sedation, amnesia, and tolerance liabilities. A prototype for such BZ ligands is imidazenil (IMD), an imidazo-benzodiazepine carboxylic acid derivative that elicits potent anticonvulsant and anxiolytic actions at doses virtually devoid of sedative, cardio-respiratory depressant and amnestic effects, and anticonvulsant tolerance liability. To define the pharmacological profile of IMD and its derivatives, we compared the anticonflict (anxiolytic), anti-proconflict (antipanic), anti-bicuculline (BIC), and maximal electroshock seizure (MES) effects, and the suppression of locomotor activity by imidazo-benzodiazepine carboxylic acid derivatives to those of DZ and bretazenil (BTZ). We report here that IMD and one of its derivatives (RO 25-2775) possess dose-dependent anticonflict, anti-proconflict, and anti-BIC actions but failed to suppress locomotor activity. Like DZ, the other IMD derivatives (enazenil, RO 25-2776, and RO 25-2847) not only elicit dose-dependent anticonflict, anti-proconflict, anti-BIC, anti-MES effects but also suppress locomotor activity. In contrast, none of the IMD derivatives studied shows any similarity to BTZ, which elicits anticonflict, anti-proconflict actions and suppresses locomotor activity but is virtually inactive against BIC-induced tonic-clonic convulsions.

Reelin is an extracellular matrix protein synthesized in cerebellar granule cells that plays an important role in Purkinje cell positioning during cerebellar development and in modulating adult synaptic function. In the cerebellum of schizophrenia (SZ) and bipolar (BP) disorder patients, there is a marked decrease ( approximately 50%) of reelin expression. In this study we measured Purkinje neuron density in the Purkinje cell layer of cerebella of 13 SZ and 17 BP disorder patients from the McLean 66 Cohort Collection, Harvard Brain Tissue Resource Center. The mean number of Purkinje neurons (linear density, neurons per millimeter) was 20% lower in SZ and BP disorder patients compared with nonpsychiatric subjects (NPS; n = 24). This decrease of Purkinje neuron linear density was unrelated to postmortem interval, pH, drugs of abuse, or to the presence, dose, or duration of antipsychotic medications. A comparative study in the cerebella of heterozygous reeler mice (HRM), in which reelin expression is down-regulated by approximately 50%, showed a significant loss in the number of Purkinje cells in HRM (10-15%) compared with age-matched (3-9 months) wild-type mice. This finding suggests that lack of reelin impairs GABAergic Purkinje neuron expression and/or positioning during cerebellar development.

Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann's area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layer I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.

Organophosphate (OP)-nerve agent poisoning may lead to prolonged epileptiform seizure activity, which can result in irreversible neuronal brain damage. A timely and effective control of seizures with pharmacological agents can minimize the secondary and long-term neuropathology that may result from this damage. Diazepam, the current anticonvulsant of choice in the management of OP poisoning, is associated with unwanted effects such as sedation, amnesia, cardio-respiratory depression, anticonvulsant tolerance, and dependence liabilities. In search for an efficacious and safer anticonvulsant benzodiazepine, we studied imidazenil, a potent anticonvulsant that is devoid of sedative action and has a low intrinsic efficacy at alpha1- but is a high efficacy positive allosteric modulator at alpha5-containing GABA(A) receptors. We compared the potency of a combination of 2mg/kg, i.p. atropine with:(a) imidazenil 0.05-0.5mg/kg i.p. or (b) equipotent anti-bicuculline doses of diazepam (0.5-5mg/kg, i.p.), against diisopropyl fluorophosphate (DFP; 1.5mg/kg, s.c.)-induced status epilepticus and its associated neuronal damage. The severity and frequency of seizure activities were determined by continuous radio telemetry recordings while the extent of neuronal damage and neuronal degeneration were assessed using the TUNEL-based cleaved DNA end-labeling technique or neuron-specific nuclear protein (NeuN)-immunolabeling and Fluoro-Jade B (FJB) staining, respectively. We report here that the combination of atropine and imidazenil is at least 10-fold more potent and longer lasting than the combination with diazepam at protecting rats from DFP-induced seizures and the associated neuronal damage or ongoing degeneration in the anterior cingulate cortex, CA1 hippocampus, and dentate gyrus. While 0.5mg/kg imidazenil effectively attenuated DFP-induced neuronal damage and the ongoing neuronal degeneration in the anterior cingulate cortex, dentate gyrus, and CA1 hippocampus, 5mg/kg or a higher dose of diazepam is required to produce similar protective effects. These finding suggests that imidazenil, a non-sedating anticonvulsant BZ ligand, is a more potent, effective, and safer drug than diazepam in protecting rats from DFP-induced seizures and the associated neuronal damage and/or ongoing neuronal degeneration.

Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes:(i) pretreatment with reversible blockers of AChE,(ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 mug/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.